Open AccessVol 10 No 4 Research article Caveolin-1 expression and stress-induced premature senescence in human intervertebral disc degeneration Sarah Kathleen Heathfield1, Christine Lyn
Trang 1Open Access
Vol 10 No 4
Research article
Caveolin-1 expression and stress-induced premature senescence
in human intervertebral disc degeneration
Sarah Kathleen Heathfield1, Christine Lyn Le Maitre2 and Judith Alison Hoyland1
1 Tissue Injury and Repair Group, Research School of Clinical and Laboratory Sciences, Faculty of Medical and Human Sciences, Stopford Building, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK
2 Biomedical Research Centre, Biosciences, Faculty of Health and Wellbeing, Sheffield Hallam University, City Campus, Howard Street, Sheffield, S1 1WB, UK
Corresponding author: Judith Alison Hoyland, judith.a.hoyland@manchester.ac.uk
Received: 20 May 2008 Revisions requested: 12 Jun 2008 Revisions received: 9 Jul 2008 Accepted: 5 Aug 2008 Published: 5 Aug 2008
Arthritis Research & Therapy 2008, 10:R87 (doi:10.1186/ar2468)
This article is online at: http://arthritis-research.com/content/10/4/R87
© 2008 Heathfield et al.; licensee BioMed Central Ltd
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Introduction Chronic and debilitating low back pain is a
common condition and a huge economic burden Many cases
are attributed to age-related degeneration of the intervertebral
disc (IVD); however, age-related degeneration appears to occur
at an accelerated rate in some individuals We have previously
demonstrated biomarkers of cellular senescence within the
human IVD and suggested a role for senescence in IVD
degeneration Senescence occurs with ageing but can also
occur prematurely in response to stress We hypothesised that
stress-induced premature senescence (SIPS) occurs within the
IVD and here we have investigated the expression and
production of caveolin-1, a protein that has been shown
previously to be upregulated in SIPS
Methods Caveolin-1 gene expression in human nucleus
pulposus (NP) cells was assessed by conventional and
quantitative real-time polymerase chain reaction (PCR), and
caveolin-1 protein expression was examined within human IVDs
using immunohistochemistry The correlation between
caveolin-1 and pcaveolin-16INK4a (biomarker of cellular senescence) gene expression was investigated using quantitative real-time PCR
Results Caveolin-1 gene expression and protein expression
were demonstrated within the human IVD for the first time NP cells from degenerate discs exhibited elevated levels of caveolin-1 which did not relate to increasing chronological age
A negative correlation was observed between gene expression for caveolin-1 and donor age, and no correlation was found between caveolin-1 protein expression and age A positive correlation was identified between gene expression of
caveolin-1 and pcaveolin-16INK4a
Conclusion Our findings are consistent with a role for
caveolin-1 in degenerative rather than age-induced changes in the NP Its expression in IVD tissue and its association with the senescent phenotype suggest that caveolin-1 and SIPS may play a prominent role in the pathogenesis of IVD degeneration
Introduction
Low back pain (LBP) is a condition that affects a significant
proportion of the population, with a lifetime incidence rate in
excess of 70% in industrialised nations [1] It not only impacts
on quality of life, but also places a substantial financial burden
on the National Health Service and the economy in general
due to loss of working days [1,2] Many cases of LBP are attributed to degeneration of the intervertebral disc (IVD) and imaging studies have indicated a link between IVD degenera-tion and LBP [3,4]
To date, no clear mechanism for IVD degeneration has been identified, although the involvement of both environmental and genetic factors has been proposed [5-8] The occurrence of
ABI = Applied Biosystems (Warrington, UK); ADAMTS = a disintegrin and metalloprotease with thrombospondin motifs; AF = annulus fibrosus; AGE
= advanced glycation endproduct; CML = carboxymethyl-lysine; Ct = cycle threshold; DMEM + F-12 = Dulbecco's modified Eagle's medium and Ham's F-12 nutrient medium; gDNA = genomic DNA; IHC = immunohistochemistry; IL = interleukin; IVD = intervertebral disc; LBP = low back pain; MMP = matrix metalloproteinase; NP = nucleus pulposus; PCR = polymerase chain reaction; PDAR = pre-developed assay reagent; PM = post mor-tem; qRT-PCR = quantitative real-time reverse transcription-polymerase chain reaction; RAGE = receptor for advanced glycation endproducts; RS
= replicative senescence; SA-β-gal = senescence-associated β-galactosidase; SD = standard deviation; SEM = standard error of the mean; SIPS = stress-induced premature senescence; TBS = Tris-buffered saline; uPAR = urokinase plasminogen activator receptor.
Trang 2IVD degeneration increases with age [9,10]; however, a
sub-set of individuals appear to exhibit accelerated degeneration
that is independent of age [5,6] This has led to speculation
that additional factors could play a key role in the development
of degeneration in some individuals
There is increasing evidence that many features of IVD
degen-eration, including altered matrix synthesis and enhanced matrix
degradation, originate at a cellular level [6,11,12] Cellular
senescence is a strong candidate for the prolonged alteration
in cellular activity observed during degeneration Senescence
and accompanying alterations in cell function have been
impli-cated in ageing-related, degenerative, and pathological
changes in a variety of tissues, including atherosclerotic
plaque development within blood vessels and osteoarthritic
alterations to cartilage [13-15] Two groups have shown
increased staining for senescence-associated
β-galactosi-dase (SA-β-gal) in cells from prolapsed and degenerate IVD
cells, respectively, when compared with non-degenerate discs
[16,17] More recently, our group has presented more
com-prehensive evidence of senescence biomarkers in human IVD
samples, demonstrating increased cellular senescence during
IVD degeneration [18] In particular, cells from degenerate
discs exhibited increased SA-β-gal activity, elevated
expres-sion of the cell cycle inhibitor p16INK4a, telomere erosion, and
a decrease in replicative potential Furthermore, a correlation
was observed between p16INK4a expression and the
expres-sion of matrix-degrading enzymes matrix metalloproteinase
(MMP)-13 and a disintegrin and metalloproteinase with
throm-bospondin motifs (ADAMTS)-5, suggesting a role for cell
senescence in the molecular processes observed during IVD
degeneration [18]
Senescence occurs naturally with ageing but can also occur
prematurely in response to stresses (such as exposure to
cytokines or oxidative stress) in a number of cell types [19-24]
Since telomeric erosion and p16INK4a protein expression are
increased in degenerate discs compared to non-degenerate
age-matched samples [18], we hypothesised that
stress-induced premature senescence (SIPS) occurs within the IVD
and may be responsible for the accelerated degeneration
observed in some individuals
Caveolae are plasma membrane compartments found
abun-dantly in terminally differentiated cells such as fibroblasts and
endothelial and muscle cells [25] The mammalian caveolin
gene family codes for three 21 to 25 kDa caveolin proteins,
which are integral membrane proteins essential for the
struc-tural integrity and function of caveolae [26] Expression of
caveolin-3 is muscle-specific, whereas caveolin-1 and
caveo-lin-2 are coexpressed in many cell types [26] Proposed
func-tions include lipid transport, membrane trafficking, and a role
in intracellular signalling pathways which stems from the
colo-calisation of caveolins with a variety of signal transduction
mol-ecules [25-28] Interestingly, caveolin-1 has been implicated
in the senescent phenotype of several cell types, including human fibroblasts, lung adenocarcinoma cells, endothelial cells, and articular chondrocytes [19,29-33] Moreover, cave-olin-1 has been proposed to mediate SIPS in murine fibrob-lasts and human articular chondrocytes in response to oxidative stress and the inflammatory cytokine interleukin-1β (IL-1β) (both of which are known to be increased during IVD degeneration) [19,31,34-38] Here, we have investigated the expression of caveolin-1 in human IVDs and correlated its expression with the cell cycle inhibitor and the biomarker of senescence p16INK4a, focusing on the nucleus pulposus (NP)
as this area shows the most evidence of cell senescence in human IVDs [18]
Materials and methods
Tissue samples
Human IVD tissue was obtained either at post mortem (PM) examination or from patients undergoing surgery, where patients were selected on the basis of magnetic resonance imaging-diagnosed degeneration and progression to anterior resection either for spinal fusion or disc replacement surgery for chronic LBP Local research ethics committee approval was obtained together with informed consent from the patient
or relatives Disc tissue was removed as detailed previously [37]
General procedure for tissue specimens
A block of tissue (incorporating annulus fibrosus [AF] and NP
in continuity) was fixed in 10% vol/vol neutral buffered formalin and embedded in paraffin wax Four micron sections were stained with haematoxylin and eosin to grade the degree of morphological degeneration according to previously pub-lished criteria that assess the demarcation between NP and
AF, proteoglycan content of the NP, presence and extent of structural fissures, and cell cluster formation [39] Potential grades range between 0 and 12 A grade of 0 to 3 indicates a histologically non-degenerate IVD, 4 to 7 indicates evidence of intermediate (or moderate) degeneration, and 8 to 12 indi-cates severe degeneration Further tissue sections were taken for immunohistochemical analysis of caveolin-1
Isolation of nucleus pulposus cells
To obtain NP cells from human IVD tissue, NP tissue was iden-tified and dissected from AF NP tissue was finely chopped and digested in a solution of 2 U/mL protease (Sigma-Aldrich, Gillingham, UK) in Dulbecco's modified Eagle's medium plus Ham's F-12 nutrient medium (DMEM + F-12) (Gibco BRL, now part of Invitrogen, Paisley, UK) for 30 minutes at 37°C NP cells were washed twice with DMEM + F-12 prior to cell iso-lation with collagenase type I treatment (0.4 mg/mL; Invitrogen)
Trang 3Conventional reverse transcription-polymerase chain
reaction
To investigate gene expression of caveolin-1 in human NP
cells, RNA was extracted from isolated cells following the
standard procedure for TRIzol® reagent (Invitrogen) cDNA
was then synthesised using Superscript II in accordance with
the instructions of the manufacturer (Invitrogen) A standard
Platinum Taq (Invitrogen) method was used for conventional
polymerase chain reaction (PCR), using a concentration of 1.5
mM MgCl2 Primers specific for caveolin-1 [19] and the
house-keeping gene 18S (Invitrogen) are detailed in Table 1 All
prim-ers were confirmed for gene specificity using BLAST (Basic
Local Alignment Search Tool) (Genbank database
sequences) Reactions, including non-template controls, were
conducted for 35 cycles, including the annealing temperature
of 58°C on a thermal cycler (MJ Research, now part of
Bio-Rad Laboratories, Hercules, CA, USA), and products were
analysed alongside a 100-base pair DNA ladder (Hyperladder
IV; Bioline, London, UK) by electrophoresis on a 1.5% wt/vol
agarose gel containing 0.2 μg/mL ethidium bromide
(Sigma-Aldrich) Product bands were visualised by UV
transillumina-tion and images were captured using Gene Snap software
(Syngene, Cambridge, UK)
Quantitative real-time polymerase chain reaction
Quantitative real-time reverse transcription-PCR (qRT-PCR)
was performed to further examine caveolin-1 gene expression
in human NP cells and to investigate any correlation between
caveolin-1 and p16INK4a gene expression in isolated NP cells
using the standard curve method of analysis as described
pre-viously [18]
Primers and probe design
Primers and FAM-MGB probe specific for human caveolin-1
were designed by Applied Biosystems (ABI) (Warrington, UK)
upon provision of caveolin-1-specific exon sequence (Gene
expression assays) (Table 1) p16INK4a primers and probe were
as described previously [18], and 18S primer/VIC-TAMRA
probe set was a pre-developed assay reagent (PDAR) pur-chased from ABI
Genomic curve standards
Genomic DNA (gDNA) was used to create standard curves for absolute quantification of copy number per reaction gDNA (Promega Corporation, Southampton, UK) was homogenised, diluted to 100 ng/μL, and sonicated on ice Serial dilutions of gDNA were prepared to generate standards with gene copy numbers of 75,000, 7,500, 750, 75, and 0 copies per 25 μL reaction
Quantitative real-time reverse transcription-polymerase chain reaction amplification
qRT-PCRs were carried out in triplicate in a 96-well plate Reactions contained 12.5 μL of mastermix (Taqman® Univer-sal PCR mastermix; ABI) and 2.5 μL of template cDNA or gDNA Primers were added to a final concentration of 900 nM and probe to a concentration of 250 nM, and molecular-grade water was added to a total reaction volume of 25 μL A gDNA standard curve for each gene was included on each plate Real-time PCR was performed using an ABI Prism 7000 sequence detection system (ABI) Reactions consisted of an initial Taq activation step of 95°C for 10 minutes to denature DNA and activate Taq polymerase followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute
Quantitative real-time reverse transcription-polymerase chain reaction analysis
Following amplification, an auto-baseline was set using the ABI 7000 sequence detection software and a threshold was set for each gene, above background levels and within the exponential phase From these, a cycle threshold (Ct) was obtained for each well and data exported into Microsoft Excel
Table 1
Details of polymerase chain reaction (PCR) primers, probes, and amplicon sizes
Conventional PCR conditions
Target Forward primer 5' to 3' Reverse primer 5' to 3' Amplicon size, base pairs (bp)
Real-time PCR primers and probes
Caveolin-1 ACT TGC AAC CGT CTG TTA TGC T FAM – ACA TGG CCC CTC CCC – MGB GCA AAG GGA TGC TTG GAT TAG GT p16 INK4a GGC TCT ACA CAA GCT TCC TTT CC FAM – ACC CTG GCT CTG ACC A –
MGB
TCA TGA CCT GCC AGA GAG AAC A PDAR, pre-developed assay reagent.
Trang 4(Microsoft Corporation, Redmond, WA, USA), where the three
Ct values for each sample were averaged Data were analysed
as described previously [18] and results were expressed as
copy number of target gene per 100 ng cDNA normalised to
18S.
Immunohistochemistry
Immunohistochemistry (IHC) was used to determine the
expression and localisation of caveolin-1 protein in the NP of
28 paraffin-embedded disc samples (Table 2) Normal human
skin tissue was used as a positive control The protocol was
based upon previously published IHC [40] Briefly, following
deparaffination, blocking of endogenous peroxidase activity,
and enzyme retrieval in 0.01% wt/vol chymotrypsin
(Sigma-Aldrich) solution at 37°C for 20 minutes, sections were
washed and incubated with 25% rabbit serum (Sigma-Aldrich)
to block non-specific binding sites Sections were then
incu-bated at 4°C overnight with mouse monoclonal antibody
against human caveolin-1 (BD Transduction Laboratories
cat-alogue number 610406, clone 2297; BD Biosciences,
Oxford, UK) (1:10 dilution in 25% rabbit serum in 0.1% bovine
serum albumin; Sigma-Aldrich) Negative control sections
were incubated with an equivalent concentration of mouse
IgG1 (Dako UK Ltd., Ely, UK) Following washes in
Tris-buff-ered saline (TBS), sections were incubated with biotinylated
rabbit anti-mouse antiserum (1:400; Dako UK Ltd.) for 30
min-utes at room temperature After further washes in TBS,
immu-noreactivity was visualised using the streptavidin-biotin
complex (Dako UK Ltd.) technique with 3,3'-diaminobenzidine
tetrahydrochloride solution (Sigma-Aldrich) Sections were
subsequently rinsed in water, counterstained with Mayer's
haematoxylin, dehydrated, and mounted with Pertex (HistoLab,
Gothenburg, Sweden)
Sections were visualised using a Leica RMDB microscope
(Leica Camera Limited, Knowlhill, Milton Keynes, UK), and
images were captured using a digital camera and Bioquant
Nova image analysis system (Bioquant Image Analysis
Corpo-ration, Nashville, TN, USA) For analysis, the NP was identified
morphologically within each disc section Within each section,
a minimum of 200 NP cells were analysed from at least five
dif-ferent fields of view and immunopositivity was calculated as a
percentage of the total cell population
Statistical analysis
Data were non-parametric and thus Mann-Whitney U tests
were conducted to compare gene copy number and numbers
of caveolin-1-immunopositive cells in non-degenerate NP
(grades 0 to 3) and degenerate NP (grades 4 to 7 and 8 to
12) Non-parametric linear regression analysis was performed
to analyse the correlation between copy numbers of different
genes and between gene copy numbers and subject age or
number of caveolin-1-immunopositive cells and subject age
Results
Caveolin-1 gene expression in human nucleus pulposus cells
cDNAs derived from cells directly extracted from the NP of 19 different IVDs, from both PM and surgical sources, were ana-lysed for expression of the caveolin-1 gene Eight samples were taken from non-degenerate IVD (grades 0 to 3; mean age
± standard deviation [SD] 45.4 ± 18.7 years) and 11 samples from degenerate IVD (grades 4 to 9; 51.7 ± 24.3 years) Gene expression for caveolin-1 was detected in the NP tissue of every sample analysed (qRT-PCR analysis) Comparison of
Table 2 Details of human nucleus pulposus samples used to study caveolin-1 protein expression by immunohistochemistry
Laboratory number Histological grade Age, years Source
PM, post mortem tissue.
Trang 5caveolin-1 gene expression by non-degenerate and
degener-ate samples demonstrdegener-ated higher gene expression in
degenerate samples (conventional RT-PCR analysis, Figure
1) This was supported by qRT-PCR analysis (Figure 2a) in
that non-degenerate samples demonstrated a median
caveo-lin-1 gene copy number of 35,220 with a range of 6,740 to
70,9222 copies per 100 ng cDNA compared with the
ele-vated degenerate median caveolin-1 gene copy number of
45,695 with a range of 7,589 to 105,626 copies per 100 ng
cDNA (Figure 2a) A negative correlation was observed
between gene expression for caveolin-1 and age of the donor
(P = 0.0472) (Figure 2b).
Immunohistochemical detection of caveolin-1 protein in
human nucleus pulposus
Caveolin-1 protein expression was investigated in 28 IVD
sam-ples (for sample details, see Table 2) Immunohistochemical
analysis for caveolin-1 demonstrated cytoplasmic/membrane
staining within the chondrocyte-like cells of the NP (Figure 3)
The percentage of immunopositive cells for caveolin-1
increased from 2.59% ± 1.01% (mean ± standard error of the
mean [SEM]) in non-degenerate discs to 13.62% ± 6.51% in
severely degenerate samples (Figure 4a) All IgG1 controls
were negative It must be noted that the majority of patients
with severely degenerate discs were above 50 years of age;
however, in the 24 samples of all grades for which the
chron-ological age of individuals was known, no correlation was
observed between caveolin-1 immunopositivity and age of the
donors (P = 0.6609) (Figure 4b).
Correlation between caveolin-1 gene expression and
Seventeen NP samples were analysed for both caveolin-1 and p16INK4a gene expression using qRT-PCR Analysis of p16INK4a expression agreed with our previous study [18] in that
a higher proportion of degenerate than non-degenerate discs expressed p16INK4a Of the five non-degenerate samples (from
PM source, mean age ± SD 45.8 ± 18.4 years), only two sam-ples expressed p16INK4a at copy numbers of 1.4 and 55.8 cop-ies per 100 ng cDNA from individuals of 30 and 75 years of age, respectively Eleven of the 12 degenerate samples (from both PM and surgical sources, 35.4 ± 12.7 years) expressed p16INK4a with median and maximum copy numbers of 32.5 and 17,075 copies per 100 ng cDNA, respectively qRT-PCR analysis demonstrated a significant correlation between cave-olin-1 and p16INK4a gene expression in the degenerate NP
samples (P = 0.02) (Figure 5).
Discussion
This study has demonstrated for the first time that cells from the NP of human IVDs express caveolin-1 and furthermore that
Figure 1
Conventional reverse transcription-polymerase chain reaction
(RT-PCR) for caveolin-1 and housekeeping gene 18S
Conventional reverse transcription-polymerase chain reaction
(RT-PCR) for caveolin-1 and housekeeping gene 18S Representative
pho-tographs following agarose gel electrophoresis of products from
con-ventional RT-PCR for caveolin-1 and 18S cDNA samples displayed are
non-degenerate samples from a post mortem (PM) source (respective
grades [G] and ages of subjects: G3, 30 years; G1, 30 years; and G2,
75 years) and degenerate samples from surgical and PM sources (G5,
29 years; G6, 34 years; and G9, 74 years) Photographs are inverted
to improve visualisation of product bands Cav-1, caveolin-1.
Figure 2
Quantitative real-time reverse transcription-polymerase chain reaction analysis of caveolin-1 gene expression levels in nucleus pulposus (NP) cells from human intervertebral disc
Quantitative real-time reverse transcription-polymerase chain reaction analysis of caveolin-1 gene expression levels in nucleus pulposus (NP)
cells from human intervertebral disc (a) Caveolin-1 gene expression
per 100 ng cDNA normalised to 18S in non-degenerate and
degener-ate NP presented as box-and-whisker plot (5–95 percentile) (b)
Corre-lation of caveolin-1 gene expression with age of subject
Non-parametric linear regression analysis (P = 0.0472; R2 = 0.2122).
Trang 6caveolin-1 gene expression and protein expression are
ele-vated in degenerate IVDs, but that this rise in caveolin-1
expression does not correlate with increasing age This is
con-sistent with a role for caveolin-1 in degenerative rather than
age-induced changes in the NP
Changes associated with tissue ageing and degeneration
have been postulated to involve cellular senescence [41-43]
Two major categories of senescence are generally described
in the literature as replicative senescence (RS) and SIPS RS
was first described by Hayflick in 1965 [44] and is widely
regarded as one of the main mechanisms underlying the
nor-mal ageing process via reduction of telomere length to critical
levels following cumulative population doublings In addition,
there are a number of reports describing premature induction
of senescence as a result of cellular exposure to stress
Fac-tors linked to the induction of SIPS vary widely, from DNA damage – for example, radiation (bovine aortic endothelial cells [45]), UV light (human fibroblasts [46] and human melanocytes [47]), chemical treatment (nasopharyngeal carci-noma cells [48] and human fibroblasts [49,50]), and oxidative stress (human fibroblasts [20,22,24] and human articular chondrocytes [19]) – to oncogenic protein overexpression (for example, ras in human fibroblasts [51]) and exposure to inflammatory cytokines such as IL-1 and tumour necrosis fac-tor-α (human chondrocytes and fibroblasts [19,21,23]) Previ-ous data from our laboratory described accelerated senescence (characterised by a variety of biomarkers, includ-ing reduced cell replication potential, elevated levels of the cell cycle inhibitor p16INK4a, increased SA-β-gal activity, and telomere erosion) in degenerate human IVDs compared with age-matched non-degenerate discs [18], suggesting that SIPS may be involved in IVD degeneration
Figure 3
Caveolin-1 immunohistochemistry
Caveolin-1 immunohistochemistry (a) Photomicrograph demonstrating
staining for caveolin-1 protein in degenerate human nucleus pulposus
(sample 28) (b) Immunoglobulin G controls were negative.
Figure 4
Analysis of caveolin-1 immunohistochemistry
Analysis of caveolin-1 immunohistochemistry (a) Percentage of cells
immunopositive for caveolin-1 protein in non-degenerate, moderately degenerate, and severely degenerate intervertebral discs Data are
shown as mean ± SEM (b) Correlation of caveolin-1 protein
expres-sion with age of subject Non-parametric linear regresexpres-sion analysis (P = 0.6609; R2 = 0.0089).
Trang 7Caveolin-1 forms homodimers, or heterodimers with its family
member caveolin-2, that insert into the plasma membrane of
terminally differentiated cells [25] The caveolin-1-rich areas
termed caveolae and the caveolin proteins themselves are
pro-posed to regulate cellular processes, including membrane
traffic, signal transduction, and cellular senescence
[25-28,52] Caveolin-1 was investigated here due to its possible
role in cellular senescence, in particular SIPS [19,31,52]
Here, we show that caveolin-1 gene expression and protein
expression are increased during IVD degeneration, but not in
a manner that is associated with increasing chronological age
Moreover, we demonstrate a correlation between caveolin-1
and p16INK4a gene expression p16INK4a is a cyclin-dependent
kinase inhibitor that prevents retinoblastoma phosphorylation
and arrests the cell cycle in the G0/G1 phase prior to entry into
the synthesis phase [53,54] Many studies have shown
increased levels of p16INK4a alongside the occurrence and
maintenance of permanent growth arrest and senescence,
including a rodent model of ageing [55-57] Previous studies
by our group and others strongly suggest a role for p16INK4a in
cellular senescence within degenerate tissue when compared
with age-matched controls [18,58] Furthermore, elevated
p16INK4a expression has been described in the premature
senescence of human fibroblasts and leukaemic cells exposed
to oncogenic ras and DNA double-strand breaks [51,59,60],
strengthening the reports that p16INK4a is a biological marker
for senescence The present study demonstrated that the
increased expression of caveolin-1 seen in the degenerate NP
positively correlated with gene expression for p16INK4a,
sug-gesting that caveolin-1 expression is linked to the senescent
phenotype observed in these cells
The literature describes evidence linking cell exposure to stressful stimuli to both caveolin-1 expression and cellular senescence In mouse NIH 3T3 fibroblasts, administration of subcytotoxic levels of H2O2 to experimentally mimic oxidative stress induced cellular senescence and increased caveolin-1 expression Treatment with H2O2 in the presence of
caveolin-1 antisense oligonucleotides reduced expression of senes-cence biomarkers, whereas transgenic overexpression of caveolin-1 induced SIPS [31] In human endothelial cells, iso-lated from atherosclerotic patients and induced to senesce, caveolin-1 expression was correlated with senescence biomarkers and with expression of 4-hydroxynonenal expres-sion (a marker of lipid peroxidation and thus oxidative stress) independently of an effect on telomere length [31] These studies strongly support a role for caveolin-1 in SIPS induced
by oxidative stress and this is further strengthened by work conducted on osteoarthritic articular chondrocytes Adminis-tration of H2O2 to these chondrocytes induced cellular senes-cence via expression of the caveolin-1 protein, a mechanism reversed by antisense oligonucleotide-mediated downregula-tion of the caveolin-1 gene [19] The same study demon-strated an identical role for the inflammatory cytokine IL-1β
Articular chondrocytes and the degenerative process observed during osteoarthritis share many characteristics with IVD cells and IVD degeneration [12,43] Interestingly, IVD cells are subjected to both oxidative stress and catabolic cytokines, which have been implicated in the induction of SIPS [19-22,24] Work published by our group suggests that IL-1β not only is increased in degenerate discs but is an important factor involved in catabolic events during IVD degeneration, including decreased matrix production and increased MMP and ADAMTS expression [37,38,61,62] Moreover, advanced glycation endproducts (AGEs) such as carboxymethyl-lysine (CML) and the receptor for AGEs (RAGE) have been localised
to the NP of degenerate IVD [34-36] CML is a tissue marker for accumulated oxidative stress [35]; therefore, its presence and that of its receptor RAGE are highly significant for both mechanisms underlying IVD degeneration and the likelihood that they could cause SIPS in human NP cells Furthermore, RAGE has been localised to caveolin-1-rich membranes in endothelial cells [63] This gives evidence, together with studies involving IL-1, that there are factors in the degenerate disc that may induce caveolin-1 expression and thus lead to the senescent phenotype described in IVD cells [16-18]
Caveolin-1-rich regions of the plasma membrane have been associated with several receptors and signalling molecules, predominantly through isolation of caveolae and colocalisation studies These studies have highlighted a subset of proteins that are relevant to IVD degeneration and to SIPS First, RAGE, described above, is known to regulate several intracellular signalling pathways, including the nuclear factor-kappa-B pathway, which is essential for the expression of MMPs present in the degenerate IVD [34,64] Second, there
Figure 5
Correlation between caveolin-1 and p16 INK4a gene expression in
degenerate nucleus pulposus samples
Correlation between caveolin-1 and p16 INK4a gene expression in
degenerate nucleus pulposus samples Caveolin-1 and p16 INK4a gene
expression (copy number per 100 ng cDNA normalised to 18S)
ana-lysed by quantitative real-time reverse transcription-polymerase chain
reaction Non-parametric linear regression analysis (P = 0.02; R2 =
0.4725).
Trang 8is evidence suggesting that caveolin-1, β1 integrin, and
uroki-nase plasminogen activator receptor (uPAR) colocalise in
human articular chodrocytes [65] uPAR has an integral role in
plasmin activation and thereby promotes catabolic events
through initiation of a proteolytic cascade through which
matrix-degrading enzymes described in IVD degeneration
such as MMPs are activated [66] Both could conceivably be
pathways via which elevated caveolin-1 levels exert aspects of
the senescent cellular phenotype observed in IVD
degeneration
Conclusion
This study has shown that caveolin-1 expression in human NP
cells is linked to IVD degeneration and is associated with the
senescent phenotype as depicted by increased expression of
p16INK4a Caveolin-1 expression was not linked to increasing
chronological age, suggesting a role in accelerated
degenera-tion which could be due to SIPS, rather than RS Further work
will elucidate the role of caveolin-1 in these related areas
Competing interests
The authors declare that they have no competing interests
Authors' contributions
SKH participated in the design of the study, performed the
majority of the laboratory work and analysis, and drafted the
manuscript CLM helped to secure funding, participated in the
design of the study and the interpretation of data, and assisted
in the preparation of the final manuscript JAH conceived the
study, secured funding, contributed to the design and
coordi-nation of the study, and participated in the interpretation of
data and extensive preparation of the final manuscript All
authors read and approved the final manuscript
Acknowledgements
This work was funded by a grant from DISCS (Diagnostic Investigation
of Spinal Conditions and Sciatica) and was undertaken in the Human
Tissue Profiling Laboratories of the Tissue Injury and Repair research
group.
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